Pectin polymers are important constituents of plant primary cell walls. They are mainly composed of chains of 1,4-linked .alpha.-D-galacturonic acid and methoxylated derivatives thereof. The use of pectin-degrading enzymes is important for the food industry, primarily in fruit and vegetable processing such as fruit juice production or wine making, where their ability to catalyse the degradation of the backbone of the pectin polymer is utilised.
Multiple pectin degrading enzymes is known to be present in various microorganisms such as Aspergillus niger. Of these, pectin methylesterase catalyses the removal of methanol from pectin, resulting in the formation of pectic acid (polygalacturonic acid). Pectate lyase cleave galacturonosyl bonds in polygalacturonic acid by .beta.-elimination, pectin lyase cleave the galacturonosyl bonds of highly methylated pectins by .beta.-elimination, and polygalacturonase hydrolyses the glycosidic linkages in the polygalacturonic acid chain.
For many purposes, it would be desirable to provide each of the pectin degrading enzymes present in, for instance, commercial preparations containing a number of different pectin degrading enzymes (an example of such a preparation is Pectinex Ultra SP.RTM., prepared from Aspergillus aculeatus, available from Novo Nordisk A/S) in a form free from other components. In this way, it would be possible to produce enzyme preparations adapted to specific purposes, such preparations either containing a single pectin degrading enzyme or arbitrary combinations thereof. To serve this end, it is convenient to provide single-component pectin degrading enzymes by recombinant DNA techniques.
Plastow et al. (1986) describe the cloning of four pectate lyase genes and one polygalacturonase gene from Erwinia in E. coli. A cloned polygalacturonase from Erwinia carotovora is described in WO 91/043 ino. U.S. Pat. No. 4,801,540 (Calgene) describes a DNA sequence encoding a tomato polygalacturonase and its expression in plants.
Behere et al. (1993), Kester and Visser (1990), Keon and Waksman (1990), Lourdes et al. (1991), and Siessere et al. (1992) disclose the characterization of polygalacturonases isolated from various fungal species including A. niger. Cloning, expression and further characterization of polygalacturonases from A. niger are described in EP 421 919 (Ciba-Geigy) and by Bussink et al. (1990), Bussink et al. (1991) and Bussink et al. (1992).